Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane

Based on electrophysiological measurements, it has been argued that the active form of cystic fibrosis trans-membrane conductance regulator (CFTR) Cl(-) channel is a multimer. It has also been demonstrated that this multimerization is likely due to PDZ domain-interacting partners. Here we demonstrate that although CFTR in vitro can self-associate into multimers, which depends on PDZ-based interactions, this may not be the case in cell membrane. Using chemical cross-linking, we demonstrated that CFTR exists as a higher order complex in cell membrane. However, this higher order complex is predominantly CFTR dimers, and the PDZ-interacting partners (Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) and NHERF2) constitute approximately 2% of this complex. Interestingly solubilizing membrane expressing CFTR in detergents such as Triton X-100, Nonidet P-40, deoxycholate, and SDS tended to destabilize the CFTR dimers and dissociate them into monomeric form. The dimerization of CFTR was tightly regulated by cAMP-dependent protein kinase-dependent phosphorylation and did not depend on the active form of the channel. In addition, the dimerization was not influenced by either the PDZ motif or its interacting partners (NHERF1 and NHERF2). We also demonstrated that other signaling-related proteins such as Gbeta and syntaxin 1A can be in this higher order complex of CFTR as well. Our studies provide a deeper understanding of how the CFTR assembly takes place in native cell membrane.     

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.     

Mass spectrometry detection of monomeric renalase in human urine

Renalase is a recently discovered secretory protein, which is suggested to play a role (which still remains elusive) in regulation of blood pressure. Earlier it was purified from urine of healthy volunteers by means of ammonium sulfate fractionation and subsequent affinity chromatography (Xu et al. (2005), J. Clin. Invest., 115, 1275). The resultant purified preparation of renalase contained 2 proteins with molecular masses of 35 and 67?C75 kDa. The authors believed that the latter represents a dimerization (aggregation) product of the 35 kDa protein. In this study we have detected relanase in urinary samples of 2 of 6 volunteers only after immunoaffinity enrichment of urinary samples subjected to ammonium sulfate precipitation. Electrophoresis of the purified preparation also demonstrated the presence of 2 proteins with molecular masses of 35 and 66 kDa, respectively. Mass spectrometry analysis of these proteins identified 35 and 66 kDa proteins as renalase and serum albumin, respectively. Thus, our results do not support suggestion on formation of renalase dimers and they indicate that urinary renalase excretion significantly varies in humans.     

Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity

Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.     

Roles of NHERF1/EBP50 in cancer

This review summarizes the emerging roles of NHERF1/EBP50 adaptor protein in tumorigenesis. NHERF1/EBP50 (Na(+)/H(+) exchanger regulating factor 1; ezrin-radixin-moesin (ERM) binding phosphoprotein of 50 kDa) is a PDZ domain-containing protein with physiological localization at the plasma membrane. We discuss in this review the functions of NHERF1/EBP50 as a linker between membrane proteins and the cytoskeleton network, as well as its involvement in different types of cancer, such as breast and liver cancers. Recent evidence obtained from our laboratory and from other groups shows that NHERF1/EBP50 is an important player in cancer progression. It appears that, depending on its subcellular distribution, NHERF1/EBP50 may behave either as a tumor suppressor, when it is localized at the plasma membrane, or as an oncogenic protein, when it is shifted to the cytoplasm. We provide here an overview of the mechanisms by which this adaptor protein controls cell transformation, and propose a model suggesting a dual role of NHERF1/EBP50 in cancer.     

RNA Dimerization Promotes PKR Dimerization and Activation

The double-stranded RNA (dsRNA)-activated protein kinase [protein kinase R (PKR)] plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15-bp dsRNA for one protein to bind and 30-bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eukaryotic initiation factor 2α, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15- to 30-bp limits: human immunodeficiency virus type 1 transactivation-responsive region (TAR) RNA, a 23-bp hairpin with three bulges that is known to dimerize. TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization to test whether RNA dimerization affects PKR dimerization and activation. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary-structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary-structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15- to 30-bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease.     

Constitutive and agonist-induced dimerizations of the P2Y1 receptor: relationship to internalization and scaffolding

In living cells, P2Y(1) receptor dimerization was quantitated by an improved version of fluorescence resonance energy transfer donor photobleaching analysis. 44% of the P2Y(1) receptors expressed in HEK293 cell membranes exist as dimers in the resting state, inducible by agonist exposure to give 85-100% dimerization. Monomer and constitutive dimers are fully active. Agonist-induced dimerization follows desensitization and is fully reversible upon withdrawal of agonist. Receptor dimers are required for internalization at 37 degrees C but are not sufficient; at 20 degrees C dimerization also occurs, but endocytosis is abolished. Removal of the C-terminal 19 amino acids abolished both dimerization and internalization, whereas full activation by agonists was retained up to a loss of 39 amino acids, confirming active monomers. This receptor is known to bind through its last four amino acids (DTSL) to a scaffolding protein, Na/H exchanger regulatory factor-2, which was endogenous here, and DTSL removal blocked constitutive dimerization specifically. Distinction should therefore be made between the following: 1) constitutive dimers tethered to a scaffolding protein, together with effector proteins, within a signaling micro-domain, and 2) free dimers in the cell membrane, which here are inducible by agonist exposure. For the class A G-protein-coupled receptors, we suggest that the percentages of free monomers, and in many cases of induced free dimers, commonly become artifactually increased; this would arise from an excess there of the receptor over its specific scaffold and from a lack of the native targeting of the receptor to that site.     

Error-prone PCR mutagenesis reveals functional domains of a bacterial transcriptional activator, TraJ

TraJ is the essential activator of P(Y), the promoter of the F and F-like plasmid tra operon that encodes the majority of the proteins for bacterial conjugation. By combining error-prone PCR mutagenesis with a two-plasmid screen, we isolated 55 missense mutations in traJ, each affecting the ability of TraJ to activate P(Y). These mutations define two distinct functional clusters (amino acids [aa] 21 to 117 and aa 150 to 219). Limited proteolytic analysis of TraJ suggested that the N- and C-terminal functional clusters are two structurally distinct domains. Most TraJ mutants exhibited decreased intracellular protein levels, and the HslVU protease-chaperone pair was found to be responsible for degrading those mutants without extracytoplasmic stress-induced overexpression. In vivo cross-linking analysis of TraJ mutants indicated that the N-terminal domain is responsible for dimerization. This was confirmed by the finding that the purified N-terminal region of TraJ forms dimers in solution. The levels of dimerization and in vivo activities of TraJ mutants are well correlated, suggesting that dimerization of TraJ is required for its biological function. We propose that the regulation of TraJ dimerization and/or its susceptibility to HslVU could be a key mechanism in various signaling processes for controlling bacterial conjugation in response to physiological or environmental stimuli.     

Na/H exchange regulatory factor 1, a novel AKT-associating protein, regulates extracellular signal-regulated kinase signaling through a B-Raf-mediated pathway

Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5'-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf.     

Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.     

Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin

Human intrinsic factor (IF) was purified from the recombinant plant Arabidopsis thaliana by affinity chromatography. Cobalamin (Cbl) saturated protein was separated by gel filtration into peaks I and II, which contained according to SDS electrophoresis the 50 kDa full-length protein IF(50) and a mixture of two fragments, respectively. Two components of peak II were identified as the 30 kDa N-terminal peptide IF(30) and the 20 kDa C-terminal glycopeptide IF(20). Measurements of M(w) under the nondenaturing conditions were conducted by static light scattering. They revealed 100 kDa IF dimers in peak I, whereas 50 kDa cleaved monomers were found in peak II. The protein devoid of Cbl dissociated to the elementary units incapable of association in the absence of Cbl. The individual proteolytic fragments bound Cbl at high concentration of the ligand; however, neither IF(30).Cbl nor IF(20).Cbl oligomerized. A mixture of two fragments IF(30) + IF(20) and Cbl produced a firm complex, IF(30+20).Cbl, which could not associate to dimers. In contrast to IF(30+20).Cbl, the saturated full-length monomers IF(50).Cbl dimerized with K(d) approximately 1 microM. We suggest a two-domain organization of the full-length protein, where two distant units, IF(30) and IF(20), can be assembled only by Cbl. They are connected by a protease-sensitive link, whose native structure is likely to be important for dimerization. However, linkage between two domains is not compulsory for Cbl binding. Advantages of the two-domain structure of IF are discussed.     

Adhesive and lateral E-cadherin dimers are mediated by the same interface

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. To study cadherin-cadherin interactions that may underlie the adhesive process, a recombinant E-cadherin lacking free sulfhydryl groups and its mutants with novel cysteines were expressed in epithelial A-431 cells. These cysteine mutants, designed according to various structural models of cadherin dimers, were constructed to reveal cadherin dimerization by the bifunctional sulfhydryl-specific cross-linker BM[PE0]3. Cross-linking experiments with the mutants containing a cysteine at strand B of their EC1 domains did show cadherin dimerization. By their properties these dimers correspond to those which have been characterized by co-immunoprecipitation assay. Under standard culture conditions the adhesive dimer is a dominant form. Calcium depletion dissociates adhesive dimers and promotes the formation of lateral dimers. Our data show that both dimers are mediated by the amino-terminal cadherin domain. Furthermore, the interfaces involved in both adhesive and lateral dimerization appear to be the same. The coexistence of the structurally identical adhesive and lateral dimers suggests some flexibility of the extracellular cadherin region.     

Phosphatidylglycerol is involved in the dimerization of photosystem II

Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1Delta3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1Delta3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two-dimensional crystals of the native dimer.     

The Na(+)/H(+) exchanger regulatory factor 2 mediates phosphorylation of serum- and glucocorticoid-induced protein kinase 1 by 3-phosphoinositide-dependent protein kinase 1

The Na(+)/H(+) exchanger regulatory factor 2 (NHERF2/TKA-1/E3KARP) contains two PSD-95/Dlg/ZO-1 (PDZ) domains which interact with the PDZ docking motif (X-(S/T)-X-(V/L)) of proteins to mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. One of the PDZ domains of NHERF2 interacts specifically with the DSLL, DSFL, and DTRL motifs present at the carboxy-termini of the 2-adrenergic receptor, the platelet-derived growth factor receptor, and the cystic fibrosis transmembrane conductance regulator, respectively. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) also carries a putative PDZ-binding motif (D-S-F-L) at its carboxy tail, implicated in the specific interaction with NHERF2. There is a 3-phosphoinositide-dependent protein kinase 1 (PDK1) interacting fragment (PIF) in the tail of NHERF2. Using pull-down assays and co-transfection experiments, we demonstrated that the DSFL tail of SGK1 interacts with the first PDZ domain of NHERF2 and the PIF of NHERF2 binds to the PIF-binding pocket of PDK1 to form an SGK1-NHERF2-PDK1 complex. Formation of the protein complex promoted the phosphorylation and activation of SGK1 by PDK1. Thus, it was suggested that NHERF2 mediates the activation and phosphorylation of SGK1 by PDK1 through its first PDZ domain and PIF motif, as a novel SGK1 activation mechanism.     

The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties

The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29-160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel beta sheet with a positively charged beta hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.     

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1)

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).     

Heat-induced dimerization of BCL-xL through alpha-helix swapping

The dimerization of anti-apoptotic BCL-xL by three-dimensional domain swapping has recently been discovered at alkaline pH; however, the high energetic barrier between the dimer and monomer forms of BCL-xL prevents them from interconverting at room temperature and neutral pH. Here, we demonstrate that BCL-xL dimers can be easily prepared by heating concentrated protein above 50 degrees C. The 38 kDa BCL-xL dimer was fully characterized by multi-resonance nuclear magnetic resonance (NMR) spectroscopy, and the mechanism of dimerization by alpha-helix swapping was confirmed. Dimerization strongly affects the NMR signals from the turn between helices alpha5 and alpha6 of BCL-xL and a portion of the long loop between helices alpha1 and alpha2. Measurements of residual dipolar couplings demonstrate that the solution structure of the BCL-xL dimer is very close to the crystal structure. Dimer formation does not prevent tight binding of ligands to the hydrophobic cleft of BCL-xL; however, binding of a BID BH3-peptide or a polyphenol drug, gossypol, to BCL-xL significantly slowed monomer-dimer interconversion and is an example of the control of BCL protein oligomerization by ligand binding.